Controlled Inhibition of Apoptosis by Photoactivatable Caspase Inhibitors

•Photocaged caspase inhibitors were evaluated for stability and turn-on of activity•A nitroindoline was superior to other photoprotecting groups•Photocaged inhibitors covalently inhibit caspases only upon irradiation•Photocaged caspase inhibitors prevent in situ apoptosis only upon light exposure Caspases control regulated cell death (apoptosis), a process that is crucial in the development of multicellular organisms as well as in various diseases. In order to spatiotemporally study apoptosis, we here develop photoactivatable caspase inhibitors. These are based on cysteine-reactive acyloxymethyl ketone electrophiles connected to a peptide targeting caspases. Importantly, the aspartate crucial for recognition by caspases is caged with a photoprotecting group. Ester photocages were found to be labile, and it was critical to have a nitroindoline cage, which forms a stable amide bond with the aspartate side chain. The nitroindoline-protected inhibitors lead to an efficient turn-on of inhibitory activity after irradiation with light. They are applicable in live cells, where they prevent anti-FAS-induced apoptosis only upon irradiation. Overall, these reagents will allow a better understanding of the spatial and temporal dimensions of apoptosis in complex, dynamic systems. Caspases control regulated cell death (apoptosis), a process that is crucial in the development of multicellular organisms as well as in various diseases. In order to spatiotemporally study apoptosis, we here develop photoactivatable caspase inhibitors. These are based on cysteine-reactive acyloxymethyl ketone electrophiles connected to a peptide targeting caspases. Importantly, the aspartate crucial for recognition by caspases is caged with a photoprotecting group. Ester photocages were found to be labile, and it was critical to have a nitroindoline cage, which forms a stable amide bond with the aspartate side chain. The nitroindoline-protected inhibitors lead to an efficient turn-on of inhibitory activity after irradiation with light. They are applicable in live cells, where they prevent anti-FAS-induced apoptosis only upon irradiation. Overall, these reagents will allow a better understanding of the spatial and temporal dimensions of apoptosis in complex, dynamic systems. Proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins and polypeptides. They are important not only in digestion and protein turnover, but also in many physiological processes such as blood coagulation, cell signaling, and apoptosis. Dysregulation of their activity is the cause of a wide variety of human diseases, and therefore proteases are attractive drug targets (Turk, 2006Turk B. Targeting proteases: successes, failures and future prospects.Nat. Rev. Drug Discov. 2006; 5: 785-799Crossref PubMed Scopus (973) Google Scholar) (Drag and Salvesen, 2010Drag M. Salvesen G.S. Emerging principles in protease-based drug discovery.Nat. Rev. Drug Discov. 2010; 9: 690-701Crossref PubMed Scopus (384) Google Scholar). Unfortunately, protease biology is highly complex for several reasons. The human genome codes for over 500 proteases, which are not all expressed at the same time and in the same place. Some proteases occur in virtually all cell types, while others are specific for certain cells (spatial dimension) or stages of development (temporal dimension). To add to the complexity, the functional role of proteases depends not only on their own expression patterns, but also on those of their substrates, which additionally vary in space and time. A further layer of complexity involves the tight regulation of protease activity: most proteases are synthesized as inactive zymogen precursors, and once activated, they are regulated by various posttranslational mechanisms. The ability to exercise a precise spatiotemporal control over dynamic protease activity would be highly desirable. This would not only enable one to target proteases at the site of disease, potentially lowering unwanted side effects that result from inhibition elsewhere in the body, but also allow one to unravel physiological functions of proteases that are difficult to study with classical methods. For example, knockouts of various caspases in mice are lethal during the embryonic or perinatal stage (Zheng et al., 1999Zheng T.S. Hunot S. Kuida K. Flavell R.A. Caspase knockouts: matters of life and death.Cell Death Differ. 1999; 6: 1043-1053Crossref PubMed Scopus (250) Google Scholar). Recently, the use of light to study biological processes has seen an increase, as light-controlled probes provide a high level of spatiotemporal precision with qualitative and quantitative control in comparison with traditional inhibitors and agonists. This field of photopharmacology includes two main strategies: cis-trans isomerization of azobenzenes (Hull et al., 2018Hull K. Morstein J. Trauner D. In vivo photopharmacology.Chem. Rev. 2018; 118: 10710-10747Crossref PubMed Scopus (260) Google Scholar) (Lerch et al., 2016Lerch M.M. Hansen M.J. van Dam G.M. Szymanski W. Feringa B.L. Emerging targets in photopharmacology.Angew. Chem. Int. Ed. 2016; 55: 10978-10999Crossref PubMed Scopus (307) Google Scholar) and photocaging (Klan et al., 2013Klan P. Solomek T. Bochet C.G. Blanc A. Givens R. Rubina M. Popik V. Kostikov A. Wirz J. Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy.Chem. Rev. 2013; 113: 119-191Crossref PubMed Scopus (966) Google Scholar) (Young and Deiters, 2007Young D.D. Deiters A. Photochemical control of biological processes.Org. Biomol. Chem. 2007; 5: 999-1005Crossref PubMed Scopus (171) Google Scholar) of essential functional groups. Although a few reports exist on cis-trans isomerization for proteasome inhibitor activation (Hansen et al., 2014Hansen M.J. Velema W.A. de Bruin G. Overkleeft H.S. Szymanski W. Feringa B.L. Proteasome inhibitors with photocontrolled activity.Chembiochem. 2014; 15: 2053-2057Crossref PubMed Scopus (38) Google Scholar) (Blanco et al., 2017Blanco B. Palasis K.A. Adwal A. Callen D.F. Abell A.D. Azobenzene-containing photoswitchable proteasome inhibitors with selective activity and cellular toxicity.Bioorg. Med. Chem. 2017; 25: 5050-5054Crossref PubMed Scopus (20) Google Scholar), the difference in inhibitory capacity of the two different forms is very modest. To date, photocaging seems a more promising approach for photoactivatable protease inhibitors. This strategy has recently been applied to cathepsin inhibitors, caged as ruthenium complexes (Respondek et al., 2014Respondek T. Sharma R. Herroon M.K. Garner R.N. Knoll J.D. Cueny E. Turro C. Podgorski I. Kodanko J.J. Inhibition of cathepsin activity in a cell-based assay by a light-activated ruthenium compound.ChemMedChem. 2014; 9: 1306-1315Crossref PubMed Scopus (49) Google Scholar) or with BODIPY-based protecting groups (Toupin et al., 2019Toupin N.P. Arora K. Shrestha P. Peterson J.A. Fischer L.J. Rajagurubandara E. Podgorski I. Winter A.H. Kodanko J.J. BODIPY-caged photoactivated inhibitors of cathepsin B flip the light switch on cancer cell apoptosis.ACS Chem. Biol. 2019; 14: 2833-2840Crossref PubMed Scopus (10) Google Scholar), leading to efficient turn-on inhibitors after irradiation. Here, we apply the concept of photopharmacology to caspase inhibitors. Members of the caspase family are essential players in apoptosis (Denault and Salvesen, 2002Denault J.B. Salvesen G.S. Caspases: keys in the ignition of cell death.Chem. Rev. 2002; 102: 4489-4500Crossref PubMed Scopus (264) Google Scholar) and other physiological processes, such as inflammation and inflammatory cell death (Van Opdenbosch and Lamkanfi, 2019Van Opdenbosch N. Lamkanfi M. Caspases in cell death, inflammation, and disease.Immunity. 2019; 50: 1352-1364Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). They are crucial for removing diseased cells as well as shaping multicellular organisms during development. Hence, precise timing and location of caspase-mediated events are of the utmost importance. In this article, we describe the design, synthesis, and evaluation of photoactivatable caspase inhibitors for application in the spatiotemporal control of apoptosis. These molecules prevent apoptosis in whole cells only upon irradiation, opening the door to functional studies of controlled cell death in space and time. Caspases have a strict requirement for a negatively charged aspartate residue in the P1 position (see Figure 1A for protease nomenclature), because of the positively charged primary recognition pocket (S1 pocket; Figure 1B). We reasoned that protection of the carboxylic acid side chain of aspartate in caspase inhibitors would prevent recognition and render an inhibitor inactive until irradiation would regenerate the free carboxylate (Figure 1C). We based our design of photocaged caspase inhibitors on peptidyl acyloxymethyl ketones (AOMKs). AOMKs are reactive warheads for cysteine proteases (Figure 1D), and their selectivity can be steered by the peptide element. In this case, we incorporated a VAD or DEVD sequence, both previously reported as favorable for caspases (Thornberry et al., 1997Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. et al.A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1805) Google Scholar) (Rano et al., 1997Rano T.A. Timkey T. Peterson E.P. Rotonda J. Nicholson D.W. Becker J.W. Chapman K.T. Thornberry N.A. A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE).Chem. Biol. 1997; 4: 149-155Abstract Full Text PDF PubMed Scopus (231) Google Scholar). Conveniently, peptidyl AOMKs can be fully synthesized on solid supports starting from properly protected chloromethyl ketone (CMK) building blocks (Kato et al., 2005aKato D. Boatright K.M. Berger A.B. Nazif T. Blum G. Ryan C. Chehade K.A.H. Salvesen G.S. Bogyo M. Activity-based probes that target diverse cysteine protease families.Nat. Chem. Biol. 2005; 1: 33-38Crossref PubMed Scopus (276) Google Scholar). The CMKs required for this study were made in solution from commercially available Fmoc-protected aspartate tert-butyl ester 1 or its isomer 3g (Scheme 1A). We started with synthesizing photocaged aspartic acids from compound 1 (Scheme 1A). Five different photoprotecting groups (PPGs) were chosen for protection of the side chain: a 2-nitrobenzyl ester (a), a more sterically hindered 2-nitrobenzyl ester (b), a 7-methoxy-coumaryl ester (c), a p-methoxy-phenacyl ester (d), and a 4-methoxy-7-nitroindoline amide (e). Compound 2a was made by esterification with nitrobenzyl alcohol, and for the sterically hindered nitrobenzyl ester (b), a one-pot Passerini reaction was utilized (Szymanski et al., 2014Szymanski W. Velema W.A. Feringa B.L. Photocaging of carboxylic acids: a modular approach.Angew. Chem. Int. Ed. 2014; 53: 8682-8686Crossref PubMed Scopus (20) Google Scholar) (Scheme 1A). Compounds 2c–d were made by nucleophilic substitution of bromo methyl derivatives of p-methoxy-phenacyl and 7-methoxycoumarin, respectively. Finally, the nitroindoline PPG was introduced starting from 4-methoxyindole, which was first reduced to 4-methoxyindoline, as described before (Hayashi et al., 2015Hayashi K. Kusaka N. Yamasaki S. Zhao Y. Nozaki H. Development of 4-methoxy-7-nitroindolinyl (MNI)-caged auxins which are extremely stable in planta.Bioorg. Med. Chem. Lett. 2015; 25: 4464-4471Crossref PubMed Scopus (6) Google Scholar), and then coupled to the aspartic acid side chain followed by regioselective nitration under the influence of silver nitrate and acetyl chloride (Scheme 1A) (Wang et al., 2009Wang X. Gao Y. Xu Y. Li L. Zhang Z. Liu J. Efficient preparation of photolabile agent MNI-glu by regioselective nitration of 4-methoxyindoline derivative.Synth. Commun. 2009; 39: 4030Crossref Scopus (7) Google Scholar). In addition, methyl-protected aspartic acid 2f was made for the synthesis of cell-permeative control inhibitors. Next, the t-Bu esters of 2a–f were cleaved, leading to side-chain-protected Asp derivatives 3a–f. Together with standard Fmoc-Asp(tBu)-OH (3g), these were then converted into CMKs 4a–g in a reported three-step one-pot sequence (Scheme 1A) (Wood et al., 2003Wood W.J. Huang L. Ellman J.A. Synthesis of a diverse library of mechanism-based cysteine protease inhibitors.J. Comb. Chem. 2003; 5: 869-880Crossref PubMed Scopus (20) Google Scholar). With the required CMK building blocks at hand, we now proceeded to the construction of photoactivatable AOMK inhibitors and unprotected control inhibitors. To this end, CMK building blocks 4a–g were loaded onto a semicarbazide resin through a hydrazone linkage (Scheme 1B). Next, the chloride was substituted by 2,6-dimethyl benzoic acid to give the desired AOMK warhead. Elongation under modified Fmoc-based solid-phase peptide synthesis conditions (Kato et al., 2005aKato D. Boatright K.M. Berger A.B. Nazif T. Blum G. Ryan C. Chehade K.A.H. Salvesen G.S. Bogyo M. Activity-based probes that target diverse cysteine protease families.Nat. Chem. Biol. 2005; 1: 33-38Crossref PubMed Scopus (276) Google Scholar) gave inhibitors 5a–b, 6a–g, 7e–g and 8e–g with either a single aspartate or a VAD or DEVD sequence (Scheme 1B). In order to apply the photocaged caspase inhibitors in cell culture as well as in potential future in vivo experiments, the inhibitors should be stable under aqueous conditions and uncage only upon irradiation. To evaluate aqueous stability, we first incubated inhibitors 5a–b, 6a and 6c–d in common buffers (HEPES and PBS) for various amounts of times and then analyzed their integrity by liquid chromatography-tandem mass spectrometry analysis. We found that PPGs with an ester linkage were prone to rapid hydrolysis (see Figures 2A–2C for 5a, Figure S1 for 5b, 6a, 6c–d). Within 20 min, more than 50% of 5a hydrolyzed to the free carboxylic acid, as detected by a retention time shift (Figure 2B) as well as mass spectrometry (Figure 2C). We found this result rather surprising, because most of these PPGs have been used in biochemical studies before, albeit in the context of different molecules. Nevertheless, aqueous lability of various phenacyl (Givens et al., 1998Givens R.S. Weber J.F. Jung A.H. Park C.H. New photoprotecting groups: desyl and p-hydroxyphenacyl phosphate and carboxylate esters.Methods Enzymol. 1998; 291: 1-29Crossref PubMed Scopus (51) Google Scholar) and coumaryl (Muralidharan et al., 2016Muralidharan S. Dirda N.D. Katz E.J. Tang C.M. Bandyopadhyay S. Kanold P.O. Kao J.P. Ncm, a photolabile group for preparation of caged molecules: synthesis and biological application.PLoS One. 2016; 11: e0163937Crossref PubMed Scopus (9) Google Scholar) (Furuta et al., 1999Furuta T. Wang S.S. Dantzker J.L. Dore T.M. Bybee W.J. Callaway E.M. Denk W. Tsien R.Y. Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis.Proc. Natl. Acad. Sci. U S A. 1999; 96: 1193-1200Crossref PubMed Scopus (519) Google Scholar) esters has been reported before. Interestingly, compound 6a hydrolyzed even faster than 5a (Figure S1). Evidently, the exact context in which these ester-protecting groups are present influences their stability, which highlights the importance of alternative carboxylic acid PPGs. Satisfyingly, the nitroindoline caged inhibitor 6e, which contains a much more stable amide linkage, did not undergo any hydrolysis up to 72 h (Figure 2A), but underwent rapid photolysis using irradiation at 365 nm (Figures 2D–2F and S1). To be biologically meaningful, caged molecules should be much less active against their targets than their uncaged counterparts in order to function as photoactivatable inhibitors. The larger the difference in activity, the easier they can be implemented in cellular assays. We therefore first determined apparent IC50 values of three nitroindoline-caged inhibitors (6e–8e) with hex-DEVD, hex-VAD, and Cbz-VAD sequences, respectively, and their uncaged forms 6g–8g (Figure 2G and Table S1). To this end, purified caspase-3 was incubated with serial dilutions of caged or uncaged control inhibitors and residual activity was measured with a fluorogenic substrate. We found that the DEVD-based inhibitor 6g was most active, with an apparent IC50 of 4.2 ± 1.2 nM. Its caged counterpart 6e displayed an IC50 of 89 ± 12 nM, resulting in a theoretical 21-fold turn-on of activity upon irradiation of the caged compound. Although for the VAD sequences, the overall activity was lower (IC50 in the submicromolar range, see Figure 2G and Table S1), the caged compounds 7e and 8e had IC50 values that were 37- and more than 150-fold worse, respectively. They therefore display a higher turn-on of inhibition than the caged inhibitor with the DEVD sequence. Based on the above IC50 values, we proceeded to in vitro inhibition experiments with a 0.1 μM concentration of the DEVD compounds and 1 μM for the VAD compounds. Indeed, incubation of caspase-3 with control inhibitors 6g–8g led to complete inhibition, as no residual enzyme activity was measured by a caspase-directed activity-based probe (Figure 2H). In contrast, no inhibition was found with photocaged compounds 6e–8e. However, upon irradiation of caged probes, no residually active caspase-3 was detected (Figure 2H), confirming that the UV-light trigger is responsible for inhibition. In order to show that photoactivatable caspase inhibition is also possible in a complex proteome, we stimulated HEK293T cell lysates with cytochrome c and dATP, as reported before (Liu et al., 1996Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4340) Google Scholar) (Berger et al., 2006Berger A.B. Witte M.D. Denault J.B. Sadaghiani A.M. Sexton K.M. Salvesen G.S. Bogyo M. Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes.Mol. Cell. 2006; 23: 509-521Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). This treatment mimics the intrinsic apoptotic pathway and efficiently activates the various caspases (Figure S2). Also here, we found that irradiated samples led to efficient caspase inhibition by VAD inhibitors 7e and 8e when used at 1 μM (Figure 2I). Note that caged DEVD inhibitor 6e already resulted in some inhibition at this concentration (Figure S2), but that careful dosing at lower concentrations resulted in inhibition only after irradiation (Figure 2J). We also checked whether caged inhibitors displayed reactivity against other proteins. To this end, we exploited click chemistry between a TAMRA-azide and the alkyne tag in photocaged probe 7e and uncaged control probe 7g. In both apoptotic and nonapoptotic cell lysates, we found that uncaged probes labeled only caspases, whereas caged probe 7e did not result in any protein labeling (Figure S2), highlighting the selectivity toward caspases only after uncaging. We finally wanted to demonstrate that the photoactivatable inhibitors can prevent the process of apoptosis in live cells only upon a light trigger. Of course, a prerequisite for photoactivation in whole cells is the absence of cellular toxicity of the used light source. We found that the UV lamps used in our studies did not lead to significant cellular toxicity as evaluated by MTT assay (Figure S3). At this point, we optimized the induction of apoptosis in human Jurkat T cells with anti-FAS antibody (Figure S3) and verified that UV irradiation alone did not rescue cells from apoptosis (Figure S3). To anticipate cell permeability issues with the negatively charged caspase inhibitors, we applied a prodrug strategy and used compounds 7f and 8f, in which carboxylic acids are temporarily protected as methyl esters, which undergo hydrolysis by intracellular esterases. For all inhibitors, we used 50 μM concentrations, as previous studies have shown that peptide-derived caspase inhibitors need high micromolar concentrations for prevention of apoptosis, most likely because of cell permeability issues (Solania et al., 2019Solania A. Gonzalez-Paez G.E. Wolan D.W. Selective and rapid cell-permeable inhibitor of human caspase-3.ACS Chem. Biol. 2019; 14: 2463-2470Crossref PubMed Scopus (8) Google Scholar). Where applicable, the incubated cells were irradiated with a handheld UV lamp directly after addition of photocaged inhibitors. Treatment of Jurkat T cells with the pan caspase inhibitor Cbz-VAD(OMe)-FMK and the AOMK control probe hex-VAD(OMe)-AOMK 7f caused complete inhibition of anti-FAS-induced apoptosis, as monitored overnight by live cell imaging using an Incucyte Zoom cell analysis system (Figure 3). In contrast, a DMSO control showed many apoptotic cells. Satisfyingly, the photocaged inhibitors hex-VAD(NI)-AOMK 7e and Cbz-VAD(NI)-AOMK 8e did not cause inhibition of apoptosis, whereas in irradiated cells, they showed up to 85%–90% inhibition of apoptosis after overnight culturing (Figures 3 and S4). Overall, this confirms that the nitroindoline-caged caspase inhibitors display cellular activity after irradiation only. In this work, we developed photoactivatable inhibitors of caspases. They consist of an AOMK-reactive electrophile and a caspase-targeting peptide, in which the critical aspartate in the P1 position is photocaged. Conveniently, these can be constructed on a solid support from the appropriate CMK building blocks. This allowed a rapid synthesis of inhibitors with various PPGs for the carboxylic acid function. One requirement for application in biologically relevant experiments is the stability under buffered, aqueous conditions. We found that ester linkages of the PPGs used here were prone to hydrolysis in the dark. Although the underlying reason for the hydrolysis is unclear, it seems that the lability is dependent on the structure of the molecule in which the PPG is incorporated. Hence, caution is needed when using ester photocages, and final molecules should be carefully analyzed for stability. Fortunately, the nitroindoline-caged caspase inhibitors, which contained an amide linkage instead of an ester, were fully stable up to 3 days. A second requirement for application in biologically relevant experiments is a large difference in the potency between caged and uncaged inhibitors. We here found turn-on of activity for the best molecule of more than 150-fold, as measured against purified caspase-3. The efficient turn-on of activity holds promise for implementation of the nitroindoline PPG in inhibitors of other proteases with a substrate specificity involving an essential carboxylic acid residue, such as carboxypeptidases (crucial C terminus), Granzyme B (crucial P1 Asp), and several members of the ADAMTS family displaying glutamyl endopeptidase activity. The photoactivatable nitroindoline derivatives developed here were able to inhibit caspases in complex, apoptotic proteomes only upon irradiation. These results also held up in cellular apoptosis, as demonstrated in a cell culture model of the extrinsic pathway of apoptosis, where efficient inhibition of cell death was achieved upon irradiation. Apoptosis is an essential process in multicellular organisms. It plays a key role in development as well as homeostasis. Caspases, which are cysteine proteases with a requirement for an aspartate in the P1 position of their substrates, are central regulators of apoptosis. They have been widely studied by using genetic methods and small-molecule tools, such as inhibitors and activity-based probes. However, these methods generally do not allow precise local and temporal control of caspase activity. Here, we designed, synthesized, and evaluated photocaged caspase inhibitors that can be activated by light. Importantly, we found that not all photocages were suitable for use in biological matrices, but that a nitroindoline-protected aspartate side chain in the P1 position is stable and leads to a high turn-on of inhibitory activity upon irradiation. We showed that these reagents can block apoptosis in cells by a specific light-triggered inhibition of caspases. Overall, they represent novel chemical tools for implementation in the study of apoptosis in space and time. Tabled 1REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesAnti-Fas Antibody (human, activating), clone CH11BIO CONNECT BVCat# 05-201; RRID: AB_309653Chemicals, Peptides, and Recombinant ProteinsFmoc-Asp(Otbu)-OH, 25gCarbolution ChemicalsCat# CC05008L-Fmoc-aspartic acid alpha-t-butyl esterFluorochemCat# 0413892-nitrobenzyl alcoholTCI EuropeCat# N0179-25g6-Nitroveratraldehyde, 96%THERMO FISHER (KANDEL) GMBHCat# A14415.06Benzyl IsocyanideTCI EUROPE NVCat# B2185-1G2-Bromo-4-methoxyacetophenone, 98%J&K ScientificCat# 329150-5G4-bromomethyl-7-methoxycoumarin, 97%ACROS ORGANICS NVCat# 329150-5G4-Methoxyindole, 98%J&K SCIENTIFIC BVBACat# 240246-5GDiazald, 100gSigma-AldrichCat# D28000-100GAc-DEVD-AMC (Caspase Fluorogenic substrate)Sanbio BVCat# 14986-5Caspase-3/7 Green Detection Reagent 25uLFisherCat# C10723Z-VAD-FMK Pan-caspase inhibitorBIO CONNECT BVCat# S7023_1mgCritical Commercial AssaysMTT Assay Kit (Cell Proliferation)AbcamCat# ab211091Pierce™ BCA Protein Assay KitThermo FisherCat# 23227TruSeq ChIP Sample Prep KitIlluminaCat# IP-202-1012Experimental Models: Cell LinesHEK293T (female)Laboratory of Molecular and Cellular Signaling, KU LeuvenN/AHuman Jurkat clone E6-1 (male)Laboratory of Molecular and Cellular Signaling, KU LeuvenN/ARecombinant DNAPlasmid: Caspase-3-His6Zhou et al., 1997Zhou Q. Snipas S. Orth K. Muzio M. Dixit V.M. Salvesen G.S. Target protease specificity of the viral serpin CrmA. Analysis of five caspases.J. Biol. Chem. 1997; 272: 7797-7800Abstract Full Text Full Text PDF PubMed Scopus (475) Google ScholarCat# 11821; RRID: Addgene-_11821Software and AlgorithmsChemDraw Professional 19.0Perkin Elmerhttps://www.perkinelmer.com/ImageJSchneider et al., 2012Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis.Nat Methods. 2012; 9: 671-675Crossref PubMed Scopus (28409) Google Scholarhttps://imagej.nih.gov/ij/Graphpad prism version 5.0GraphPad Software Inc.https://www.graphpad.com/scientific-software/prism/Adobe Illustrator CS4 version 14Adobehttps://www.adobe.com/be_en/products/illustrator.htmlAdobe photoshop CS4 version 11.0Adobehttps://www.adobe.com/be_en/creativecloud/education-plans.html?mv=search&sdid=8DN85NTM Open table in a new tab Further information and requests for resources and reagents may be directed to and will be fulfilled by the Lead Contact, Steven Verhelst ( [email protected] ) All unique/stable reagents generated in this study are available from the Lead Contact without restrictions. This study did not generate/analyze any datasets/code. Human Jurkat clone E6-1 (male) cells were grown in RPMI media (R0883-500ML Sigma) supplemented with 10% FBS, 2 mM glutamax, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. They were maintained at 37°C with 5% CO2 at a cell concentration between 1 × 105 and 1 × 106 viable cells/mL. Fresh medium was added or replaced every 2 to 3 days. HEK293T (female) cells were grown in DMEM media (D6546-500ML Sigma) supplemented with 10% FBS, 2 mM glutamax, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. They were maintained at 37°C with 5% CO2. Cells were split every 3 to 4 days according to ATCC protocol. 0.1 mg/ml solutions of probes 6e-8e were prepared in 20 mM HEPES buffer (pH 7.4). They were then irradiated with UV light at 366 nm and aliquots from various time points (0, 1, 2, 5 and 10 min) were analyzed by LC-MS. The graphs were analyzed by LabSolutions software version 5.82 SP1. Area under the curve of peaks in the chromatogram were integrated the sum of caged and uncaged inhibitors was normalized to 100% and graphed in GraphPad Prism 5.02. For the test of stability against hydrolysis, the probes 5a-b, 6a, 6c-6e were incubated with 20 mM HEPES (pH 7.4) or PBS buffer in the dark and aliquots from various time points were analyzed by LC-MS. Integration was done as described above. Human Jurkat clone E6-1 cells were seeded in 96-well plates at a density of 2.5×104 cells/100 μL/well. Duplicates were irradiated with either 2 min, 5 min, 10 min, or no irradiation with a 100-watt, 365 nm longwave UVA lamp. The plate was centrifuged at 1000X G, 4°C for 5 mins and he media was discarded. A mixture of 50 μl serum-free media and 50 μl MTT reagent (ab211091) was added. The plate was incubated at 37 °C for 3 h followed by addition of 150 μl MTT solvent into each well. The plate was shaken before reading the absorbance at 590 nm in a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices). Recombinant, His6-tagged caspase-3 (Addgene: 11821), (Zhou et al., 1997Zhou Q. Snipas S. Orth K. Muzio M. Dixit V.M. Salvesen G.S. Target protease specificity of the viral serpin CrmA. Analysis of five caspases.J. Biol. Chem. 1997; 272: 7797-7800Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) was expressed and purified as described before (Stennicke and Salvesen, 1999Stennicke H.R. Salvesen G.S. Caspases: preparation and characterization.Methods. 1999; 17: 313-319Crossref PubMed Scopus (157) Google Scholar). The IC50 of the inhibitors was performed on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices) using 96-well black plates with 340 nm excitation and 475 nm emission. The Ac-DEVD-AMC substrate final concentration used was 50 μM and Cas-3 final concentration